Stem cells (SCs) are natural units of tissue repair and homeostasis. Their versatility holds promise for tissue regeneration, particularly for less controversial adult SCs. Adult epithelial SCs of the skin reside within the innermost (basal) layer of the interfollicular epidermis (IFE) as well as the bulge, a niche located within the hair follicle (HF). Basal IFE SCs function in normal homeostasis to form the stratified epithelial barrier of terminally differentiated cells that are constantly shed from the body surface. HF-SCs function in homeostasis to fuel hair growth and the cyclical bouts of regeneration and degeneration of the HF. During injury, HF-SCs participate with basal IFE SCs to repair damaged epidermis and sebaceous glands. Both basal IFE SCs and HF-SCs can be cultured and propagated long-term outside their native niches under conditions where their stemlike features are retained. This remarkable feature of keratinocyte SCs renders them fundamentally important to a range of clinical applications and issues, ranging from gene therapy to burn operations, hair replacement and treatment of blindness arising from corneal degeneration. In the past grant period, we developed a strategy for marking & purifying the slow-cycling populations of adult murine SCs from their natural niches. In the last period, we focused on HF label retaining cells (LRCs), showing that they possess properties of SCs and undergo self-renewal, replenish HFs during cycling, and repair both sebaceous glands and IFE in response to injury/loss of their resident progenitors. We showed that HF-SCs express a group of transcription factors-- Sox9, Lhx2, NFATc1 & Tcf3/4--which basal IFE SCs do not, and we used these markers to trace the origins of HF-SCs to embryogenesis. We also showed that Sox9 not only marks these early SCs but also is essential for their maintenance as HF-SCs both in vivo and in vitro. We also showed that in contrast to adult HF-SCs, which only transiently repair epidermal wounds, neonatal HF-SCs provide robust and long-term wound repair. This is particularly intriguing in that during aging, the resting phase of HFs increases, and the IFE becomes thinner and loses proliferative potential. Our newfound ability to track and monitor SCs over time now affords a segue into exploring the molecular basis for the age-related decline in SC potential. In addition, our findings raise the following key questions: (1). What are the molecular differences between neonatal and adult HF-SCs and how can they explain mechanistically the biological differences in HF-SCs that occur during aging? (2) Are the adult SCs within the IFE basal layer also slow-cycling as postulated 3 decades ago, or are these cells a reflection of the aging process? What are the molecular characteristics of basal IFE cells and what are their embryonic origins? How do basal IFE SCs differ from HF-SCs and are these differences reflected in different potentials for tissue regeneration and long-term self-renewal? (3) Are the differences that define basal IFE and HF-SCs intrinsic or extrinsic? Both neonatal SC populations can be cultured and propagated in vitro--do they maintain their molecular and tissue regenerative differences in vitro or do the cultured cells converge upon a common program of gene expression and tissue regenerative potential? (4) Of the 1% of the mouse genome whose expression is preferentially up/downregulated in HF-SCs or basal IFE SCs in vivo and in vitro, which are the genes that are functionally important to these features of SCs? What genes regulate self-renewal and/or long term potential? What maintains SCs in their undifferentiated state? Can we exploit the information gained by optimizing the ability of skin epithelial SCs to be maintained long term in culture without losing features of their stemness? By addressing these issues, we expect to uncover new insights into understanding how skin SCs possess and maintain their proliferative/tissue regenerative potential and are kept in an undifferentiated state until mobilized to generate tissue. This research is prerequisite to ascertaining the potential of skin SCs for regenerative therapies that go beyond burn treatments. PUBLIC HEALTH RELEVANCE: Stem cells are natural units of tissue repair and homeostasis, and their versatility holds promise for tissue regeneration. This research focuses on deciphering the properties of embryonic and adult skin stem cells that exist within the basal layer of the epidermis and the bulge of the hair follicle. We will focus on delineating the intrinsic mechanisms that render skin SCs their ability to self-renew in vitro and in vivo and to select one of three differentiation pathways: epidermis, hair follicle and sebaceous gland. This study is a fundamental prerequisite to ascertaining the potential of skin stem cells for regenerative therapies that go beyond burn treatments.